Psyche: A Journal of Entomology

Psyche: A Journal of Entomology / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 4036571 |

Utako Kurosu, Shigeyuki Aoki, Keigo Uematsu, Mayako Kutsukake, Takema Fukatsu, "Defensive Nymphs of the Woolly Aphid Thoracaphis kashifolia (Hemiptera) on the Oak Quercus glauca", Psyche: A Journal of Entomology, vol. 2016, Article ID 4036571, 11 pages, 2016.

Defensive Nymphs of the Woolly Aphid Thoracaphis kashifolia (Hemiptera) on the Oak Quercus glauca

Academic Editor: G. Wilson Fernandes
Received12 Mar 2016
Revised02 Aug 2016
Accepted16 Aug 2016
Published05 Oct 2016


Aphid nymphs with enlarged fore- and mid-legs were found from woolly colonies of Thoracaphis kashifolia (Hormaphidinae, Nipponaphidini) on leaves of the evergreen Quercus glauca in Japan. It was shown that they grasped an introduced moth larva with their legs and some inserted their stylets deep into the body. These defenders were first-instar nymphs of the alate generation and were produced by aleyrodiform apterae from early September onward. There was a large variation in the size of their forelegs. First-instar nymphs (to be alates) produced early in the season had fore-femorotrochanters shorter than those produced later. The molting rate (the percentage of pharate individuals) of the latter was very low (less than 5% to zero), suggesting their semisterility. Although first-instar nymphs with various lengths of forelegs joined to attack moth larvae, these facts indicate that an incipient caste differentiation occurs within the first-instar nymphs of the alate generation.

1. Introduction

Since the discovery of aphid soldiers in Colophina clematis [1], sterile or nonsterile defensive nymphs have been found in many species of two aphid subfamilies, Eriosomatinae and Hormaphidinae [25]. The former subfamily consists of three tribes, Eriosomatini, Pemphigini, and Fordini, and the latter also consists of three, Hormaphidini, Cerataphidini, and Nipponaphidini [6, 7]. These aphid species basically have a host-alternating life cycle; most of them induce galls on their primary host and form exposed colonies on their secondary host. Defensive individuals (usually first- or second-instar nymphs [2] but at times fourth-instar nymphs [8] or apterous adults [9, 10]) have been recorded from the gall or the primary-host generations of all six tribes [917], and from the secondary-host generations of Eriosomatini [1], Cerataphidini [15, 1821], and Hormaphidini [22]. Although exules of Paracletus cimiciformis (Fordini) have recently been shown to suck on ant larva hemolymph [23], defensive behavior on the secondary host has been unknown from the remaining three tribes to date.

In the course of studying the life cycle of the aphid Thoracaphis kashifolia (Nipponaphidini) in Japan, we noticed soldier-like first-instar nymphs with thickened fore- and mid-legs in its woolly colonies on leaves of the evergreen oak Quercus glauca. Having introduced lepidopteran larvae onto the colonies, we succeeded in inducing defensive behavior by these nymphs. In this paper, we describe the life cycle of T. kashifolia, the defensive behavior of the first-instar nymphs and when they are produced in the life cycle. Because there was a large variation in the size of their forelegs, we address the issue whether soldier-caste differentiation occurs in this species.

2. Materials and Methods

2.1. Study Organism

The aphid Thoracaphis kashifolia (Uye) (the species epithet has been misspelled as “kashifoliae” [6, 24, 25] or “kashiwae” [2628]) forms colonies on the upper surfaces of leaves of Quercus glauca (Figures 1(a) and 1(b)) in the south-western half of Japan [27, 28] and in Taiwan [24]. A record from Q. acuta [29] remains to be confirmed. The apterous adults are aleyrodiform (Figures 1(c) and 2(a)), sessile, and flattened and are found throughout the year on the leaves [27]. The colonies produce alates in autumn and become remarkably woolly like candy floss (Figure 1(b)) during this period. The life cycle is supposed to be anholocyclic, without returning to Distylium racemosum [6, 27], which is the only known primary host of Nipponaphidini in Japan [3032]. In this paper, colonies that are covered with long wax filaments (Figure 1(b)) are called “woolly colonies,” while those that are not are “ordinary colonies” (colonies in Figure 1(a) but the central woolly colony).

2.2. Sampling of the Aphids

To investigate the annual life cycle and the colony composition, T. kashifolia was sampled from Quercus glauca in various months. We regarded the aphids on a single leaf as a colony and sampled colonies mainly in Tama, Hachioji and Ome, western Tokyo, Japan, in 2013–2016. Additional colonies were sampled in Tsuchiura (Ibaraki Prefecture), Kashihara (Nara Prefecture), and Kyoto (Kyoto Prefecture) in 2015. The whole colonies each were preserved in a 30 mL glass vial of 80% ethanol together with the leaf. Later, under a dissecting microscope in the laboratory, the aphids in 23 woolly colonies listed in Table 1 and 22 ordinary colonies in Table 2 were detached from the leaf, counted, and sorted into the following groups: (1) apterous adults, (2) first-instar nymphs to be apterae, (3) non-first-instar nymphs to be apterae, (4) alate adults, (5) first-instar nymphs to be alates, and (6) non-first-instar nymphs to be alates.

Colony codeCollection placeDate of collectionNumber of nymphs to be alates% first-instar nymphs to be alatesMost advanced stage of the alate generation

15226Tama, Tokyo4 Sep. 2015616 (722)90.2 (27.5)2nd instar
15228Tama, Tokyo12 Sep. 2015174 (265)99.3 (16.8)2nd instar
15230Tama, Tokyo20 Sep. 2015252 (315)61.1 (9.1)3rd instar
Exp#1Tama, Tokyo28 Sep. 2015 (29 Sep)425 (532)32.9 (1.4)4th instar
Exp#2Tama, Tokyo28 Sep. 2015 (29 Sep)922 (1150)40.6 (2.3)4th instar
Exp#3Tama, Tokyo28 Sep. 2015 (29 Sep)460 (532)27.4 (0.8)4th instar
Exp#4Tama, Tokyo30 Sep. 2015 (1 Oct)427 (549)35.4 (1.3)Adult
Exp#5Tama, Tokyo30 Sep. 2015 (1 Oct)1203 (1483)37.8 (1.8)Adult
Exp#6Tama, Tokyo30 Sep. 2015 (1 Oct)442 (568)42.8 (4.8)4th instar
Exp#7Tama, Tokyo30 Sep. 2015 (1 Oct)548 (651)40.9 (2.2)4th instar
15241Kyoto, Kyoto Pref.5 Oct. 2015160 (257)31.3 (2.0)Adult
14158Tama, Tokyo8 Oct. 2014 (10 Oct)218 (377)11.0 (0)Adult
14160Tama, Tokyo8 Oct. 2014 (10 Oct)240 (618)56.7 (0)Adult
15257Tama, Tokyo13 Oct. 2015210 (255)21.9 (4.3)4th instar††
15258Tama, Tokyo13 Oct. 2015258 (371)24.0 (0)Adult
15260Tama, Tokyo13 Oct. 2015172 (267)25.0 (2.3)Adult
UE1013Tsuchiura, Ibaraki Pref.13 Oct. 201588 (180)1.1 (0)Adult
14167Hachioji, Tokyo26 Oct. 201481 (254)6.2 (0)Adult
14168Hachioji, Tokyo26 Oct. 201425 (92)0Adult
15271Tama, Tokyo27 Oct. 201585 (165)1.2 (0)Adult
15272Tama, Tokyo27 Oct. 201543 (112)0Adult
15273Tama, Tokyo27 Oct. 201574 (198)1.4 (0)Adult
15274Tama, Tokyo27 Oct. 201576 (157)2.6 (0)Adult

Date of fixation in parenthesis. Colony size in parenthesis. Percentage to all nymphs to be alates, with the molting rate (%) of first-instar nymphs in parentheses. Estimated from a subsample. ††Alate adults had probably been produced in this colony.

Colony codeCollection placeDate of collectionNumber of apterous adultsNumber of nymphs to be apteraeNumber of nymphs to be alates

15001Tama, Tokyo3 Jan, 2015720
15003Tama, Tokyo3 Jan, 2015350
16004Tama, Tokyo8 Feb, 20165260
16006Tama, Tokyo8 Feb, 20161310
15022Tama, Tokyo3 Mar, 20151820
15119Tama, Tokyo21 Apr, 2015640
15144Kashihara, Nara Pref.19 May 20157420
15208Tama, Tokyo17 Jun, 201530310
15218Tama, Tokyo27 Jul, 201556170
15221Tama, Tokyo19 Aug, 20156410
15223Tama, Tokyo19 Aug, 20159510
15236Tama, Tokyo30 Sep, 2015060
15237Tama, Tokyo30 Sep, 2015040
15238Tama, Tokyo30 Sep, 20150250
15239Tama, Tokyo30 Sep, 20150230
14170Tama, Tokyo26 Oct, 20141520
14171Tama, Tokyo26 Oct, 20140190
13163Ome, Tokyo6 Nov, 2013030
13164Ome, Tokyo6 Nov, 2013060
15322Tama, Tokyo16 Dec, 20153330
15323Tama, Tokyo16 Dec, 201510840
15324Tama, Tokyo16 Dec, 20151360

A teneral aptera.First-instar nymph(s) only.

From some woolly colonies, emerged alates were collected and used for transfer experiments to confirm whether these alates are sexuparae or secondary migrants (Section 2.5). For the same purpose, some alates (22 alates collected in Tama on 30 September 2015, nine in Tama on 15 October 2014, and 12 in Ome on 6 November 2013) were confined, together with a piece of paper, in a 5 mL cotton-plugged glass vial to force their larviposition there. A few days later, after confirming first-instar nymphs walking in the vial, 80% ethanol was poured into it. These nymphs were slide-mounted (see Section 2.3), and it was determined whether they were of sexuals or virginoparae (i.e., whether their mothers were sexuparae or secondary migrants). To supplement this, ten alates from colony 15241 and ten alates from colony UE1013 (Table 1) were slide-mounted, and it was determined whether the embryos in their bodies were the same in morphology as the first-instar nymphs born in the glass vial.

2.3. Examination of Aphid Morphology

For slide preparation, aphids preserved in 80% ethanol were cleared in heated 10% KOH solution. These aphids were stained with either Evans’ blue or acid fuchsine, dehydrated in a mixture of glacial acetic acid and methyl salicylate for one day, and mounted in balsam via a mixture of xylol–phenol and pure xylol. Many slide-mounted specimens were examined under a light microscope. Since there seemed to be large variation in the sizes of their fore- and mid-legs, all or about 120–180 subsampled first-instar nymphs to be alates from the 23 woolly colonies listed in Table 1 were slide-mounted, and one of their fore-femorotrochanters was measured. For the first-instar nymphs in one colony (Exp#6 in Table 1), the length and width of one fore-femorotrochanter (defined in Figure 4(a)) were measured. Measurements were made using a digital camera (FX630; Olympus, Tokyo, Japan) equipped with image analysis software (FlvFs; Flovel, Tachikawa, Japan). It was also recorded whether these first-instar nymphs had the next instar cuticle developing inside (i.e., whether they were in the pharate stage). The percentage of nymphs in the pharate stage roughly corresponds to the degree of their sterility; if the defensive nymphs were completely sterile and did never molt, no nymphs with the next instar cuticle would be found. All non-first-instar nymphs to be alates in two colonies (Exp#1 and 14160 in Table 1) were also slide-mounted to know the composition of instars and whether they were in the pharate stage.

2.4. Defensive Test

To confirm whether aphid nymphs with enlarged legs would really attack other insects, the following experiment was carried out. A total of seven leaves with woolly colonies, which seemed to have produced defensive nymphs, were carefully removed from trees of Quercus glauca in Tama on 28 and 30 September 2015. They were each kept in a plastic container with a sheet of paper, and on the day of collection, two lepidopteran larvae (collected from leaves of Broussonetia kazinoki; ca. 3–8 mm) were placed on each colony. When an introduced larva was attacked by aphids within a short period, the larva was placed on a sheet of paper and the attacking behavior was recorded by a video camera attached to a dissecting microscope. After taking a video, the larva and aphid nymphs clinging to it were deposited in a vial of 80% ethanol. When introduced larvae were not attacked by aphids, they were left in the container under a room temperature for approximately 24 hours and examined and deposited in 80% ethanol together with aphids clinging to them. Nine out of 11 larvae attacked by defensive nymphs were macerated in 10% KOH solution, stained with acid fuchsine, and mounted on a glass slide together with the nymphs to examine whether the nymphs really pierced the larval skins under a light microscope. Aphid nymphs that attacked the remaining two larvae were slide-mounted after being detached from the larvae. The remaining aphids were preserved in vials of 80% ethanol, and later defensive first-instar nymphs and other morphs were sorted and counted under a dissecting microscope as mentioned in Section 2.2.

2.5. Transfer Experiment

To confirm whether alates of Thoracaphis kashifolia are secondary migrants (alate virginoparae), a transfer experiment was carried out with a test tree (ca. 2.5 m tall, 5 cm in diameter at 50 cm height) of Quercus glauca planted in a garden in Tama. The tree was free from T. kashifolia. Eight colonies of T. kashifolia were collected from other trees of Q. glauca in Tama and Hachioji on 13 October 2015 and kept in plastic containers. Many alates emerged from these colonies. Five twigs of the test tree were chosen and covered with a nylon bag (ca. 50 × 90 cm), and a total of 570 alates were put into the five bags on 14 and 18 October. All leaves in the five bags were examined on 24 October 2015, 22 November 2015, and 7 February 2016.

2.6. Data Analysis

The comparison of fore-femorotrochanteric length between attacking and non-attacking first-instar nymphs was performed by t-test, after checking the data for normal distribution (Kolmogorov–Smirnov test) and homogeneity of variance (F-test). Differences in the molting rate between the first instar and the remaining three instars were analyzed using Fisher’s exact test with Holm’s correction for multiple comparisons. Differences in the molting rate between first-instar nymphs with longer forelegs and those with shorter forelegs within three colonies (15226, 15228, and 15230) were analyzed by median test with Fisher’s exact test. For the relationships between the fixation date of colonies (collected in Tama in 2015) and the proportion of molting first-instar nymphs, we used a generalized linear model with binomial errors. All statistical analyses were performed with the software R v3.2.3 [33].

3. Results

3.1. Occurrence of Defensive Nymphs

Colony size and composition of our samples are listed in Tables 1 and 2. Ordinary colonies, or colonies that were not covered with long wax filaments, contained only one kind of first-instar nymphs which were small and had ordinary fore- and mid-legs and short marginal setae on the abdomen (Figure 2(b)). Such colonies were seen throughout the year (Table 2), and there is no doubt that these first-instar nymphs would grow to sessile apterae (Figures 1(c) and 2(a)). Colonies covered with long wax filaments, or woolly colonies (Figures 1(a) and 1(b)), were seen from September to early November. In these colonies (except those collected late in the season) there were first-instar nymphs of another kind: they were larger than first-instar nymphs to be apterae and had enlarged fore- and mid-legs with large, strongly curved claws, and long marginal setae on the abdomen (Figure 3(a)). Our analysis of colony composition revealed that these first-instar nymphs would grow to alates through wing-padded nymphal stages and that the first- to fourth-instar nymphs to be alates excreted long wax filaments that made the entire colony like candy floss (Figure 1(b)). Defensive nymphs were first-instar nymphs to be alates (see below).

3.2. Defensive Behavior

In all seven colonies onto which two lepidopteran larvae were introduced, one or both larvae were attacked by defensive nymphs (Figure 1(d), Table 3). Three of the 14 larvae were found hidden under the paper sheet and seemed to have escaped from being attacked by aphid nymphs, while the remaining eleven were attacked by one to 60 nymphs (115 in total). Four nymphs were detached from the larva after being deposited in ethanol, but the remaining 111 were still tightly clinging to the larva. All 115 nymphs were first-instar nymphs to be alates and in the nonpharate stage. It was observed under a dissecting microscope that defensive nymphs were tightly clinging to the lepidopteran larvae with their enlarged mid- and forelegs and seemed to pierce them with their stylets. When attacked, the larvae twisted their bodies and bit off parts of some nymphs clinging to the larvae with their mouthparts (supplementary video 1 in Supplementary Material available online at Examination of the slide-mounted specimens of nine lepidopteran larvae attacked by aphid nymphs revealed that the stylets of 11 attacking nymphs were inserted in the bodies of the larvae (Figure 5). The maximum length of the stylets inserted in the larvae was 170 μm; it was approximately as long as a fore-femorotrochanter of a small defensive nymph. The seven colonies also contained 17–76 (mean 39.3) first-instar nymphs to be apterae, but none of them joined to attack the lepidopteran larvae.

Colony codeLepidopteran larva: codeLepidopteran larva: length in mmDate and time of introductionDate and time of confirmationNumber of aphids that attacked the larva

Exp#11a528 Sep., 16:0029 Sep., 15:002
Exp#11b428 Sep., 16:0029 Sep., 15:000
Exp#22a528 Sep., 16:0028 Sep., 16:051
Exp#22b528 Sep., 16:0029 Sep., 16:009
Exp#33a428 Sep., 16:0028 Sep., 17:308
Exp#33b628 Sep., 16:0029 Sep., 15:005
Exp#44a430 Sep., 15:501 Oct., 15:002
Exp#44b330 Sep., 15:501 Oct., 15:005 ()
Exp#55a5 30 Sep., 15:501 Oct., 15:000
Exp#55b630 Sep., 15:501 Oct., 15:0019
Exp#66a730 Sep., 15:5030 Sep., 16:5060 ()
Exp#66b?30 Sep., 15:501 Oct., 15:002
Exp#77a330 Sep., 15:501 Oct., 18:002
Exp#77b830 Sep., 15:501 Oct., 18:000

Number of aphids that were detached from the larva after being deposited in alcohol is in parenthesis.
3.3. Size and Molting Rate of Defensive Nymphs

There was a large variation in the size of fore- and mid-legs of first-instar nymphs to be alates. Some had distinctly enlarged fore- and mid-legs (Figures 3(a), 4(a), and 4(b)) like Colophina soldiers, while some had only slightly enlarged or nearly ordinary legs (Figures 3(b) and 4(c)) and others were intermediate between the two. The length and width of one fore-femorotrochanter are shown as a scattered diagram for almost all first-instar nymphs in a colony (Exp#6) used in the defensive test: 31 nymphs to be apterae and 186 nymphs to be alates including 62 that actually attacked the introduced moth larvae (Figure 6). The 62 nymphs that attacked the larvae had forelegs of various sizes (Figure 6) and their fore-femorotrochanters were not longer than those of the remaining 124 nymphs that did not join the attack (t-test, ).

First-instar nymphs to be alates in three colonies sampled early in the season (on 4, 12, and 20 September) had forelegs that were shorter than those sampled later in the season (Figure 7; see also Figure 8), and their “molting rates” (sensu Akimoto [11]), or the proportions of those nymphs that had the second-instar cuticle developing inside, were high, 27.5, 16.8, and 9.1%, respectively (Table 1). On the other hand, first-instar nymphs in the seven experimental colonies (colonies Exp#1–7) collected near the end of September and the colonies collected thereafter showed small values of the molting rate, less than 5%, and some (e.g., colonies 14158, 14160, and 15258) even zero percent (Table 1). Picking up 16 colonies collected in Tama in 2015 from Table 1, the molting rate of first-instar nymphs decreased with the date of fixation (generalized linear model with binomial errors, , ). The molting rate of first-instar nymphs in colonies sampled near the end of September and thereafter, colonies Exp#1 and 14160, for example, was also lower when compared with the second- to the fourth-instar nymphs (Figure 9). (Note that in colony Exp#1, alate adults had not yet appeared. Because the colony was relatively young, the molting rate of the fourth-instar nymphs was low.)

In the early-sampled three colonies (for colony 15228, see Figure 8(a), left), first-instar nymphs with shorter forelegs tended to have the next instar cuticle developing inside (median test with Fisher’s exact test; ). In colonies sampled later in the season, the molting rate was so low that the tendency could not be confirmed statistically; however, defensive nymphs with large forelegs molt at least at times. In colony 15257 (sampled on 13 October 2015), two out of 46 first-instar nymphs to be alates had the next-instar cuticle; their fore-femorotrochanters were 217 and 216 μm long, the fifth and sixth longest in that colony.

3.4. Results of the Transfer Experiment

Offspring of the alates introduced into the five bags colonized on a few leaves in four bags. When examined on 22 November 2015, a total of 22 aphids (2 apterous adults and 20 nymphs to be apterae) were seen on the upper sides of seven leaves of Quercus glauca. On 7 February 2016, 15 apterous adults and one (presumably dead) nymph were found on four leaves. Although, taking the number of the mother alates (more than 500) into consideration, the success rate in colonization may be low, the result shows that the alates are not sexuparae but secondary migrants. All first-instar nymphs born in the glass vials were to be apterae, or of the same phenotype as in Figure 2(b). The ten alates (from colony 15241) collected in Kyoto and the ten alates (from colony UE1013) collected in Tsuchiura also turned out to be secondary migrants. No sexupara was found in this study.

3.5. Predators Found in the Field

Colonies of Thoracaphis kashifolia were rather free of predators. From the 23 sampled woolly colonies and the 22 ordinary colonies we found no predator but a few dead syrphid eggs; it is not certain whether they were killed by defensive nymphs. Only one predator we found from woolly colonies was a hemerobiid larva (ca. 9 mm long). When found (in Tama on 14 October 2015), it was still alive but three defensive nymphs were tightly clinging to near the tip of its abdomen and one clinging to its thorax, near the base of its left mid-leg. We also found a chrysopid larva (ca. 10 mm long) from an ordinary colony, with a dead apterous adult of T. kashifolia on its back (in Tama on 19 August 2015). No ants were seen attending woolly or ordinary colonies.

4. Discussion

4.1. Convergent and Peculiar Features of the Defensive Nymph

As described in Section 3.2, first-instar defensive nymphs of Thoracaphis kashifolia clasp a predator with their enlarged fore- and mid-legs and pierce it with their stylets. Such attacking behavior is also known in soldiers of the eriosomatine Colophina clematis [1]. Their thickened legs with large, strongly curved claws (Figures 4(a) and 4(b)) are similar to those of the soldiers of Colophina spp. in shape (cf. [34, 35]), which is no doubt due to convergent evolution. On the other hand, the defensive nymphs of T. kashifolia have an idiosyncratic attacking device. The aphids of the tribe Nipponaphidini on the secondary host have long stylets, which are far longer than the rostrum [36], like adelgids [37]. When not extended, the stylets are kept coiled in the head; it is not easy to measure how long they are. In one slide-mounted defensive nymph, the stylets were extended about 330 μm from the apex of the rostrum. Defensive nymphs often inserted their stylets deep in the body of a lepidopteran larva to which they were clinging; the length of the stylets inserted in the larva was up to 170 μm (Section 3.2). Such deep insertion of stylets has been unknown from defensive nymphs of other groups. Defensive nymphs of T. kashifolia may use the stylets inserted in the body of an enemy as an anchor, so as not to be detached easily from it.

Another peculiar feature of the defensive nymph of T. kashifolia is its eyes. An aphid nymph of Hormaphidinae has a pair of triommatidia, each of which consists of three facets. In the first-instar nymphs of T. kashifolia (both nymphs to be apterae and to be alates), one facet is located apart from the remaining two, on the underside of the head and directed downward (Figure 4(d)). The same type of triommatidia is known in the first-instar nymphs of Metathoracaphis isensis [31], which also forms colonies on the upper sides of leaves of the host oak (Quercus gilva). This suggests that such triommatidia may be an adaptation in the life on the upper side of a leaf and that the downward-directed facets might enable the nymphs living on the upper side to perceive enemies on the underside through the leaf tissue.

4.2. Soldier-Caste Differentiation?

Two morphologically distinct phenotypes of first-instar nymphs occur in Thoracaphis kashifolia: first-instar nymphs to be apterae (Figure 2(b)) and those to be alates (Figure 3). The former are clearly smaller than the latter as exemplified in Figure 6. Such dimorphism also occurs in the nipponaphidine Neothoracaphis quercicola [38], N. yanonis and N. saramaoensis (our unpublished results), and Reticulaphis sp. [39], all of which produce tiny, flattened apterae on leaves and may have evolved in association with the miniaturization and/or flattening of their aleyrodiform apterae. Coexistence of two morphologically distinct generations in a colony where first-instar nymphs of one generation play a defensive role is already known in the gall-forming aphid Pemphigus spyrothecae [40, 41]. The issue we address below is whether caste differentiation occurs within the first-instar nymphs of the alate generation, and not between the apterous and alate generations.

As mentioned in Section 3.3, first-instar nymphs produced early in September had smaller fore- and mid-legs than those produced later. Also, their molting rate was higher than the latter. These facts indicate that a kind of soldier-caste differentiation occurs in the alate generation of T. kashifolia; first-instar nymphs as shown in Figure 3(a) are soldiers and those in Figure 3(b) are to be reproductives. However, there were many first-instar nymphs with forelegs of intermediate sizes (Figure 7), and nymphs with forelegs of various sizes attacked lepidopteran larvae (Figure 6). In addition, although the molting rate of first-instar nymphs produced late in the season was extremely low (from less than 5% to zero), some still tried to molt to the next instar. The demarcation between the two castes is therefore not clear cut. This is an incipient kind of soldier differentiation, or colonies of T. kashifolia are at an intermediate state between those with sterile soldiers (as in Colophina clematis [1]) and those with “monomorphic” defensive nymphs which all are supposed to have the potential to grow into reproductives (as in Hemipodaphis persimilis [11] or Hamamelistes spp. [13, 42]).

4.3. Life Cycle of Thoracaphis kashifolia in Japan

The present study confirmed that the life cycle of Thoracaphis kashifolia is anholocyclic in Japan, as had been suggested by Takahashi [27]. That is, aleyrodiform apterous adults propagate themselves by parthenogenesis throughout the year on leaves of the oak Quercus glauca. They give birth to first-instar nymphs that grow to alates from early September. These alates are secondary migrants; they migrate to trees of Quercus glauca. First-instar nymphs of the alate generation play a defensive role. During October, the number of the alate generation decreases steeply. The seven colonies used in the defensive test (fixed on 29 September and 1 October) contained 425–1203 (mean 632) nymphs to be alates, while six colonies sampled on 26 and 27 October contained only 25–85 (mean 64) such nymphs (Table 1); the number of nymphs decreased to approximately 1/10 during 25–28 days. The number of first-instar nymphs to be alates decreased even more steeply, to 6/1000. This steep decrease could not be explained by molting of the first-instar nymphs, because the molting rate during this period was very low (Table 1, Figure 9). It was no doubt due to the high mortality of the defensive nymphs in exposed colonies.

We still do not know whether or how often a new colony of T. kashifolia begins with a single aphid. Our preliminary (unpublished) observations showed that some new colonies were formed on leaves near a woolly colony by more than one first-instar nymph to be aptera (e.g., colonies 15236–9 in Table 2). Although new colonies will also be formed by alates (Section 3.4), we have not yet confirmed this type of colony founding under natural conditions.

5. Conclusion

In this paper we made it clear that an incipient soldier-caste differentiation occurs in the alate generation of the aphid Thoracaphis kashifolia. This is the first discovery of defensive individuals from Nipponaphidini on their secondary hosts. The discovery was a little surprise because apterous adults of this group are sessile and seem to be protected by the hard exoskeleton. The defensive individuals are first-instar nymphs of the alate generation and therefore are produced only when aphids of the alate generation are present.

Competing Interests

The authors have no affiliations or involvement with any organization that has a financial interest in the results discussed in this paper.


The authors thank Haruo Kinuura and Furumi Komai for their help in collecting the materials. Ken Ohtsu kindly edited the supplementary video. This study was in part supported by Chuo University Leave Program for Special Research Projects (to Utako Kurosu in fiscal year 2015). Keigo Uematsu was supported by a Research Fellowship of JSPS for Young Scientists.

Supplementary Materials

Two moth larvae being attacked by a few defensive nymphs of Thoracaphis kashifolia in an experiment with the colony Exp#3. The first moth larva is trying to remove, with its mouthparts, an aphid nymph which is tightly clinging to its body. A defensive nymph on the second moth larva (from 41 seconds on) is still moving its legs; its right hind leg is lacking (probably it was bitten off by the moth larva). The video was taken in the laboratory on 28 and 29 September 2015.

  1. Supplementary Material


  1. S. Aoki, “Colophina clematis (Homoptera, Pemphigidae), an aphid species with ‘soldiers’,” Kontyû, vol. 45, no. 2, pp. 276–282, 1977. View at: Google Scholar
  2. D. L. Stern and W. A. Foster, “The evolution of soldiers in aphids,” Biological Reviews of the Cambridge Philosophical Society, vol. 71, no. 1, pp. 27–79, 1996. View at: Publisher Site | Google Scholar
  3. S. Aoki, “Soldiers, altruistic dispersal and its consequences for aphid societies,” in Genes, Behavior and Evolution of Social Insects, T. Kikuchi, N. Azuma, and S. Higashi, Eds., pp. 201–215, Hokkaido University Press, Sapporo, Japan, 2003. View at: Google Scholar
  4. N. Pike and W. A. Foster, “The ecology of altruism in a clonal insect,” in Ecology of Social Evolution, J. Korb and J. Heinze, Eds., pp. 37–56, Springer, Berlin, Germany, 2008. View at: Google Scholar
  5. P. Abbot, “The physiology and genomics of social transitions in aphids,” in Advances in Insect Physiology: Genomics, Physiology and Behaviour of Social Insects, A. Zayed and C. F. Kent, Eds., vol. 48, chapter 5, pp. 163–188, Academic Press, London, UK, 2015. View at: Publisher Site | Google Scholar
  6. R. L. Blackman and V. F. Eastop, Aphids on the World's Trees: An Identification and Information Guide, CAB International, Wallingford, UK, 1994.
  7. L. Podsiadlowski, “Phylogeny of the aphids,” in Biology and Ecology of Aphids, A. Vilcinskas, Ed., pp. 1–13, CRC Press, Boca Raton, Fla, USA, 2016. View at: Google Scholar
  8. S. Aoki, U. Kurosu, and C. D. von Dohlen, “Colony defense by wingpadded nymphs in Grylloprociphilus imbricator (Hemiptera: Aphididae),” Florida Entomologist, vol. 84, no. 3, pp. 431–434, 2001. View at: Publisher Site | Google Scholar
  9. M. Inbar, “Competition, territoriality and maternal defense in a gall-forming aphid,” Ethology Ecology & Evolution, vol. 10, no. 2, pp. 159–170, 1998. View at: Publisher Site | Google Scholar
  10. K. Uematsu, M. Kutsukake, T. Fukatsu, M. Shimada, and H. Shibao, “Altruistic colony defense by menopausal female insects,” Current Biology, vol. 20, no. 13, pp. 1182–1186, 2010. View at: Publisher Site | Google Scholar
  11. S. Akimoto, “Shift in life-history strategy from reproduction to defense with colony age in the galling aphid Hemipodaphis persimilis producing defensive first-instar larvae,” Researches on Population Ecology, vol. 34, no. 2, pp. 359–372, 1992. View at: Publisher Site | Google Scholar
  12. W. A. Foster, “Experimental evidence for effective and altruistic colony defence against natural predators by soldiers of the gall-forming aphid Pemphigus spyrothecae (Hemiptera: Pemphigidae),” Behavioral Ecology and Sociobiology, vol. 27, no. 6, pp. 421–430, 1990. View at: Publisher Site | Google Scholar
  13. H. Shibao, M. Shimada, and T. Fukatsu, “Defensive behavior and life history strategy of the galling aphid Hamamelistes kagamii (Homoptera: Aphididae: Hormaphidinae),” Sociobiology, vol. 55, no. 1, pp. 117–132, 2010. View at: Google Scholar
  14. M. Kutsukake, H. Shibao, N. Nikoh et al., “Venomous protease of aphid soldier for colony defense,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 31, pp. 11338–11343, 2004. View at: Publisher Site | Google Scholar
  15. S. Aoki and U. Kurosu, “A review of the biology of Cerataphidini (Hemiptera, Aphididae, Hormaphidinae), focusing mainly on their life cycles, gall formation, and soldiers,” Psyche, vol. 2010, Article ID 380351, 34 pages, 2010. View at: Publisher Site | Google Scholar
  16. T. Fukatsu, A. Sarjiya, and H. Shibao, “Soldier caste with morphological and reproductive division in the aphid tribe Nipponaphidini,” Insectes Sociaux, vol. 52, no. 2, pp. 132–138, 2005. View at: Publisher Site | Google Scholar
  17. S. P. Lawson, A. W. Legan, C. Graham, and P. Abbot, “Comparative phenotyping across a social transition in aphids,” Animal Behaviour, vol. 96, pp. 117–125, 2014. View at: Publisher Site | Google Scholar
  18. S. Aoki, U. Kurosu, and W. Sirikajornjaru, “A new soldier-producing aphid species, Pseudoregma baenzigeri, sp. nov., from northern Thailand,” Journal of Insect Science, vol. 7, article 38, 10 pages, 2007. View at: Publisher Site | Google Scholar
  19. A. W. Shingleton and W. A. Foster, “Behaviour, morphology and the division of labour in two soldier-producing aphids,” Animal Behaviour, vol. 62, no. 4, pp. 671–679, 2001. View at: Publisher Site | Google Scholar
  20. S. Aoki, U. Kurosu, M. Kutsukake et al., “The aphid Ceratovacuna nekoashi (Hemiptera: Aphididae: Hormaphidinae) and its allied species in Korea, Japan and Taiwan,” Entomological Science, vol. 16, no. 2, pp. 203–221, 2013. View at: Publisher Site | Google Scholar
  21. M. Hattori, T. Yamamoto, and T. Itino, “Clonal composition of colonies of a eusocial aphid, Ceratovacuna japonica,” Sociobiology, vol. 62, no. 1, pp. 116–119, 2015. View at: Google Scholar
  22. S. Akimoto, K. Ozaki, and Y. Matsumoto, “Production of first-instar defenders by the Hormaphidid gall-forming aphid Hamamelistes cristafoliae living anholocyclically on Betula maximowicziana,” Japanese Journal of Entomology, vol. 64, no. 4, pp. 879–888, 1996. View at: Google Scholar
  23. A. Salazar, B. Fürstenau, C. Quero et al., “Aggressive mimicry coexists with mutualism in an aphid,” Proceedings of the National Academy of Sciences of the United States of America, vol. 112, no. 4, pp. 1101–1106, 2015. View at: Publisher Site | Google Scholar
  24. R. Takahashi, “Additions to the aphid fauna of Formosa,” Philippine Journal of Science, vol. 48, no. 1, pp. 69–73, 1932. View at: Google Scholar
  25. G. Remaudière and M. Remaudière, Catalogue of the World's Aphididae, Institut National de la Recherche Agronomique, Versailles, France, 1997.
  26. O. Shinji, Monograph of Japanese Aphididae, Shinkyo Sha Shoin, Tokyo, Japan, 1941 (Japanese).
  27. R. Takahashi, “Three new genera of Aphididae from Japan (Homoptera),” Kontyû, vol. 26, no. 4, pp. 181–186, 1958. View at: Google Scholar
  28. M. Sorin, “Physiological and morphological studies on the suction mechanism of plant juice by aphids,” Bulletin of University of Osaka Prefecture, Series B, vol. 18, pp. 95–137, 1966 (Japanese). View at: Google Scholar
  29. K. Uye, “Notes on three species of Pemphigidae from Kyushu,” Insect World, vol. 28, no. 1, pp. 14–15, no. 3, pp. 92–94, no. 4, pp. 127–128, 1924 (Japanese). View at: Google Scholar
  30. M. Sorin, “The aphids causing galls on Distylium racemosum in Japan,” in Population Structure, Genetics and Taxonomy of Aphids and Thysanoptera, J. Holman, J. Pelikán, A. F. G. Dixon, and L. Weismann, Eds., pp. 219–223, SPB Academic Publishing, The Hague, The Netherlands, 1987. View at: Google Scholar
  31. M. Sorin, “Three new species and a new subspecies of Aphididae (Homoptera) causing galls on Distylium racemosum from Japan,” Bulletin of Kogakkan University, vol. 35, pp. 235–260, 1996. View at: Google Scholar
  32. S. Aoki, M. Kutsukake, U. Kurosu, H.-T. Yeh, M. Sano, and T. Fukatsu, “Nipponaphis species (Aphididae: Hormaphidinae) that form green galls on Distylium racemosum in Japan,” Entomological Science, vol. 18, no. 4, pp. 420–434, 2015. View at: Publisher Site | Google Scholar
  33. R Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2015.
  34. S. Aoki, “Occurrence of dimorphism in the first instar larva of Colophina clematis (Homoptera, Aphidoidea),” Kontyû, vol. 44, no. 2, pp. 130–137, 1976. View at: Google Scholar
  35. S. Aoki, “A new species of Colophina (Homoptera, Aphidoidea) with soldiers,” Kontyû, vol. 45, no. 3, pp. 333–337, 1977. View at: Google Scholar
  36. D. Noordam, “Hormaphidinae from Java (Homoptera: Aphididae),” The Zoologische Verhandelingen, Leiden, vol. 270, pp. 1–525, 1991. View at: Google Scholar
  37. R. F. Young, K. S. Shields, and G. P. Berlyn, “Hemlock woolly adelgid (Homoptera: Adelgidae): stylet bundle insertion and feeding sites,” Annals of the Entomological Society of America, vol. 88, no. 6, pp. 827–835, 1995. View at: Publisher Site | Google Scholar
  38. R. Takahashi, “Aphididae of Formosa, Part III,” Report of Department of Agriculture, Government Research Institute of Formosa, no. 10, pp. 1–121, 1924. View at: Google Scholar
  39. E. C. Zimmerman, Insects of Hawaii, Volume 5, Homoptera: Sternorhyncha, University of Hawaii Press, Honolulu, Hawaii, USA, 1948.
  40. S. Aoki and U. Kurosu, “Soldiers of a European gall aphid, Pemphigus spyrothecae (Homoptera: Aphidoidea): why do they molt?” Journal of Ethology, vol. 4, no. 2, pp. 97–104, 1986. View at: Publisher Site | Google Scholar
  41. N. Pike, C. Braendle, and W. A. Foster, “Seasonal extension of the soldier instar as a route to increased defence investment in the social aphid Pemphigus spyrothecae,” Ecological Entomology, vol. 29, no. 1, pp. 89–95, 2004. View at: Publisher Site | Google Scholar
  42. H. Shibao and T. Fukatsu, “Altruistic defenders in a gall-forming aphid of the tribe Hormaphidini (Homoptera, Aphididae, Hormaphidinae) on its primary host,” Insectes Sociaux, vol. 50, no. 2, pp. 167–173, 2003. View at: Publisher Site | Google Scholar

Copyright © 2016 Utako Kurosu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly as possible. We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. Review articles are excluded from this waiver policy. Sign up here as a reviewer to help fast-track new submissions.